System and method of flow cytometry and sample handling

ABSTRACT

An improved nozzle system for a flow cytometer and accompanying methods have been invented for a high efficiency orientation and sorting process of a flat sample and dedicates items such as equine or bovine sperm cells. This improved nozzle system comprises a nozzle with a novel interior surface geometry that can both gently accelerate the cells and can include an elliptical-like, single torsional interior surface element within the nozzle, i.e., a single torsional orientation nozzle. The elliptical-like, single torsional interior surface element may have a laminar flow surface and may produce the simplest flow path for applying minimal forces which act in either an accelerative nature or orienting hydrodynamic forces, namely, the single torsional orientation forces, to orient a flat sample such as animal sperm cells into a proper direction for an analyzing and efficiently sorting process in clinical use, for research and for the animal insemination industry.

[0001] This invention relates to an improved nozzle apparatus for a flowcytometer system and methods for improving flow cytometry. Specifically,this invention relates to a novel design of a nozzle interior surfacegeometry that gently handles and orients a sample into a proper radialdirection for analyzing and efficient sorting. The invention alsofocuses on systems for sorting delicate cells, especially living spermcells.

BACKGROUND OF THE INVENTION

[0002] Flow cytometers have been in clinical and research use for manyyears and their applications in animal industry such as animal breedingindustry has been rapidly increasing. A commercially available flowcytometer typically utilizes a cylindrical fluid geometry in its nozzle.This type of flow cytometer system has a focusing flow path withsymmetry of revolution, as described in some U.S. Patents (U.S. Pat.Nos. 5,602,039, 5,483,469, 4,660,971, 4,988,619 and 5,466,572). Thistype of design, according to the law of similarity, does not produceradially oriented samples. In clinical, animal breeding, and biologicalresearch fields, when cells such as sperm cells are sorted, they may bepre-stained with a dye that produces fluorescence when exposed to anexcitation light source. As was explained in U.S. Pat. No. 5,135,759 toLawrence Johnson, a flow cytometer that detects the emitted fluorescenceperpendicular to the axis of flow can be used with high precision in themeasurement and discrimination of the DNA content of the cells. However,as others have noted, even this precision in measuring the DNA contentcan only be achieved most efficiently when the cells of interest arespherical or cylindrical (Dean et al., 1978, Biophys. J. 23: 1-5). Asfor sperm cells—which have flattened heads—the observed fluorescenceintensity depends largely upon the proper orientation of the heads withrespect to the detector. Sperm cells emit a stronger fluorescent signalfrom the edge than the flat surface Therefore, the intensity of thefluorescent signal is dependent on the orientation of the sperm head asit passes the detector. Because DNA content is determined byfluorescence and because fluorescent intensity is affected byorientation, DNA content determination can be compounded by lack oforientation in a nozzle. For this reason, without radial orientation,the resulting fluorescence intensity distribution obtained for normal,randomly oriented sperm heads reflects both DNA content and headorientation. Because the cells emit a brighter fluorescence signal fromthe head edge (Gledhill et al., 1976, J. Cell Physiol. 87: 367-376;Pinkel et al., 1982, Cytometry 7: 268-273) the accuracy of DNA contentdetermination (which may differ by as little as 3.5%) is highly affectedby the cells orientation. For this reason, the conventional flowcytometer has experienced limitations, especially when sorting flattenedsperm cells or other non spherical or non-cylindrical cells and thelike.

[0003] Additionally, certain cells can exhibit decreased functionalityas a result of the sort process. This can be particularly true for cellssuch as mammalian sperm cells which are not only mechanically delicate,but also which can become functionally impaired (as perhaps seen throughreduced fertility) or even mortally wounded as a result of someoccurrence in the sort process. For flow cytometry efforts with delicatecells there have been significant limitations on abilities. This is mostacute in the highly specialized field of sperm cell sorting not onlybecause the cells themselves are unusually delicate, but also becausethere is a need for extremely high sorting rates for physiological andpractical reasons. These two competing needs have proven to poseuniquely critical challenges in the unique field of sperm sorting forcommercial breeding purposes. Thus, while these two aspects—gentlehandling and orientation —are perhaps independently applicable to avariety of instances, in many instances they can act synergistically.Both their independent characters and their synergistic interrelationsare perhaps most acute in the commercial sperm sorting field.Interestingly, this synergy and potential interrelationship appears notto have been fully appreciated prior to the present invention.

[0004] Viewed in isolation, the aspect of proper orientation of a samplecontaining particles or cells can thus be seen to play an important rolein the flow cytometer signal intensity and quality and in sortingefficiency. Efforts to hydrodynamically orient the sample have been madeand the use of hydrodynamic orientation of the sample in flow throughsystems and flow cytometers have been explored in last few decades(Fulwyler, 1977, J. Histochem. Cytochem. 25: 781-783; Kachel et al.,1977, J. Histochem. Cytochem. 25: 774-780; Dean et al., supra).Hydrodynamic orientation of the sample within the flow cytometer canenhance precise measurement of relative DNA-stain content and can alsoprovide a potentially useful measurement of morphological parameterssuch as cell thickness and degree of curvature of the flat face. Forsome applications, this orientation is straightforward. However, whendelicate cells (such as sperm cells) or other particles are involved,however, a more gentle technique has been necessary. For example, asample injection tube with a wedge shaped tip has even been used in someefforts to increase percentage of the oriented cells (Dean et al., 1978,Biophys. J. 23: 1-5; Fulwyler, 1977, J. Histochem. Cytochem. 25:781-783; Johnson et al., 1986, Cytometry 7: 268-273; Pinkel et al.,1982, Cytometry 3: 1-9; Welch et al., 1994, Cytometry 17 (suppl. 7):74). Because of the wedge shaped tip of the sample injection tube, thesample stream tended to be drawn into a thin ribbon by the sheath fluidas opposed to a cylindrical stream. Cells with flat heads such asmammalian sperm, often encountered the sheath fluid at a higher speed(100 mm/sec), and were then rotated so that their flat sides were in theplane of the ribbon. Unfortunately, the separation of the orientationevent and the ultimate analysis event can cause less than optimalresults. Therefore, this technique has not been practically shown to beas advantageous as desired.

[0005] In a different application, Kachel and his colleagues (Kachel etal., supra) demonstrated the law of similarity and discussed three typesof flow paths that influenced the moving particles. They concluded that,to achieve uniform radial orientation with hydrodynamic forces for flatparticles such as flattened red blood cells, the preferred flow pathwould be the one whereby unilateral constriction can be obtained. Themost simple flow path that exhibits an increased unilateral constrictionin use with a flow through system would be made of a tube with anellipsoidal cross section, and would also end in an ellipsoidal outlet.In one arrangement, the long axis of this ellipsoidal outlet would belocated at a right angle to the long axis in the cross section of theconstricting elliptical tube. However, since the elliptical outlet doesnot produce the type of droplets desired for a high speed flow cytometercell sorter, this arrangement was not intended to be used in, and hasapparently not been applied to, a flow cytometer.

[0006] In a similar effort, Rens and his colleagues designed a nozzletip that had an elliptical interior and an elliptical exit orifice (Renset al., 1998, PCT Publication No. PCT/US98/15403; Rens et al., 1998,Cytometry 33: 476-481; Rens et al., 1999, Mol. Reprod. Dev. 52: 50-56).This interior contained a first ellipsoidal zone and a secondellipsoidal zone that were separated by a transitional zone. All thezones each had a long axis and a short axis. The long axis of the secondellipsoidal zone was oriented 90° to that of the first ellipsoidal zone.A cylindrical orifice, drilled through a jewel, was located at the endof the ellipsoidal exit orifice and served as the final exit. Thisdevice partially solved the problem of random orientation as existed ina conventional flow cytometer and could orient about 60% of the totalflattened sperm cells from a boar each time through the flow cytometer.Nevertheless, when hydrodynamic forces in a flow path were taken intoconsideration, flat particles passing through the nozzle designed byRens and his colleagues have received unnecessary stresses. For delicatecells, and especially for the perhaps more delicate sperm cells such asequine or bovine sperm cells, this approach simply did not appear toyield the desired efficiency either in orientation or in cell viability.

[0007] Thus, there existed a long felt but unsatisfied need for theinvention while the needed implementing arts and elements had long beenavailable. This need concerned the ability to gently handle and perhapsorient the particles or cells to be analyzed, the ability to properlyanalyze and sort efficiently, and the ability to minimize thepotentially stressful situation that the flow cytometer caused for theparticles or cells. Further, while problems existed in conventional flowcytometers, a full appreciation that a problem existed and what theproblem was theretofore unseen by those skilled in the art. Substantialattempts by those skilled in the art to fill the need or to cope withthe difficulties had been made but had not been fully successful mostlikely because of a failure to understand what exactly the problems wereand perhaps how they interrelated. Some efforts made by those skilled inthe art even matured into patents which seemed to have touched on theproblems but, in fact, they tended in some regards to teach away fromthe technical direction in which the present inventors went.

SUMMARY OF THE INVENTION

[0008] It is therefore the object to present an improved nozzle interiorsurface geometry that produces the simplest flow path for applyingnecessary hydrodynamic forces to accelerate and perhaps orient a sampleinto a proper direction for analyzing and efficient sorting purposes.This improved nozzle interior surface geometry can comprise either orboth of: an appropriately configured accelerative force feature and/oran elliptical-like, single torsional interior surface element within asingle torsional orientation nozzle that produces the specialhydrodynamic forces, namely, single torsional orientation forces.

[0009] As the present invention now shows, the problems with undesirablecell stress could be viewed as at least in part due to eitherinappropriate handling forces, specifically: inappropriate accelerativeforces, or the existence of a second torsional force created by thesecond ellipsoidal zone. As to the accelerative forces applied, devicesoften utilized abrupt transitions internal to the nozzle and so causedextreme acceleration over short distances. As to the orientation aspect,for example, approaches such as that of Ren (mentioned earlier) showed,that after cells had been oriented by a first torsional force created bya first ellipsoidal zone, an additional—perhaps doubling—stress wasapplied. Specifically, the flat particles were already in an orientedposition after they were oriented from a random position by the firstellipsoidal zone. They were ready to exit in oriented positions. At thistime, however, the devices of Rens and others unnecessarily twistedthese oriented flat particles a second time by the hydrodynamic forcescreated by a second ellipsoidal zone. As the present invention shows,these designs are not fully efficient in a high speed flow cytometer.When flat sperm cells with tails are oriented through this type of thenozzle, besides its inefficiency, the geometry in this type of thenozzle apparently impacts twice the torsional forces. This appears tounnecessarily and highly stress or damage the long tailed sperm cellsbefore they exit the nozzle. In addition, in some designs where theorifice is made of a jewel that is separate from the main interior, asmooth laminar flow can also be affected to some degree. This couldcause almost instantaneous acceleration and so could unnecessarilystress the cells and could affect the orientation of the alreadyoriented sperm cells. Therefore, the approaches of Rens and other morerecent efforts actually teach away from the more efficient, lessaccelerative and less torsional and smooth laminar interior surface ofan embodiment of the present invention.

[0010] It is another object to design the simplest nozzle interiorsurface geometry that provides laminar flow surface and at the same timethat reduces the distortion of the sample, especially of sperm cells.

[0011] Yet another object is to present a system which can more quicklyand more accurately measure and sort the sample, especially delicatesperm cells in research and clinical use and in the animal inseminationindustry.

[0012] A further object to provide methods for improving orientation andsorting efficiency of the sample, especially the sperm cells in the flowcytometry for research and clinical use and animal inseminationindustry.

[0013] Naturally, further objects of the invention are disclosedthroughout other areas of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a cross sectional view of a portion of a flow cytometershowing a sheath fluid container, a sample injection tube and a nozzleof the present invention. This figure also shows a relative location ofthe sample tube within the nozzle.

[0015]FIG. 2 is a three-dimensional view of one nozzle tip and itsrelative position with the sheath fluid container (here the nozzle body)with a sample injection tube and a nozzle tip. FIG. 2A is a schematicdrawing of the sample injection tube that has a beveled tip and acircular mouth.

[0016]FIGS. 3A, 3B, and 3C are schematic drawings of one of the presentembodiments of the nozzle. FIG. 3A is a three-dimensional view of thenozzle tip showing the first ellipticity-increasing zone, the desiredellipse demarcation location, the ellipticity-decreasing zone, theconical zone, the cylindrical zone, and the circular exit orifice. FIG.3B is a schematic cross sectional view showing the tapered interiorsurface of the nozzle in a unitary design. FIG. 3C is a cross sectionalview of the cylindrical zone and the circular exit orifice.

[0017]FIG. 4A is a bottom view of the nozzle tip region showingspecifically the circular exit orifice. FIG. 4B is a top view of theinterior design of the nozzle showing the largest circular mouth, thedesired ellipse demarcation location, the larger circular mouth of theconical zone and the smallest circular mouth of the cylindrical zone.The diameter of the smallest mouth is also that of the circular exitorifice.

[0018]FIG. 5 shows how the single torsional orientation nozzle works inorienting flat particles.

[0019]FIG. 6 is a schematic diagram of an example of a nozzle havingaxial motion surfaces as may have existed in the prior art.

[0020]FIGS. 7a, 7 b, and 7 c are plots of the theoretical axialvelocity, acceleration, and rate of change of acceleration motions withrespect to location as may exist for a nozzle such as that shownschematically in FIG. 6.

[0021]FIG. 8 is a schematic diagram of an example of a nozzle havingaxial motion surfaces according to one embodiment of the presentinvention.

[0022]FIGS. 9a, 9 b, and 9 c are plots of the theoretical axialvelocity, acceleration, and rate of change of acceleration motions withrespect to location as may exist for a nozzle such as that shownschematically in FIG. 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] As can be seen from the illustrations and in keeping with theobjects of the present invention, the basic concepts of the presentinvention can be implemented in different ways. Referring to FIG. 1, itshows a portion of a flow cytometer system where a sample is processedinto individual droplets prior to being analyzed and sorted. As is wellunderstood by those having ordinary skill in the art, from the schematiccross sectional view, a sheath fluid container (1) may contain somesheath fluid (2) imported through a sheath fluid port (not shown). Asample injection system comprises a sample injection tube (3) connectedto a sample reservoir (not shown). The sample injection system generallyacts to provide the appropriate flow of some sample material to a nozzlesystem. The sheath fluid container at the same time introduces a sheathfluid into the nozzle system. The sample may be surrounded by the sheathfluid to form a sample-containing fluid and may then exit the nozzlesystem through a drop-forming mechanism through which thesample-containing fluid forms small droplets. These small droplets maypass through a free falling area at a high speed of over about 20 metersper second by combination of oscillation by an oscillator and highpressure from the flow cytometer system. Subsequently, these smalldroplets, i.e., the sample-containing drops, may be analyzed by ananalytical system (not shown) at the free falling area. If living cellssuch as flat sperm cells are introduced as the sample material, they maybe stained with one or more fluorescent dyes. These sperm cells may becarried in single file in the sheath fluid stream past the analyticalsystem (not shown). The analytical system may include a focused laserwhose wavelength is adjusted to excite a fluorescent dye that may bepresent. The fluorescence signal collected from each cell can then besensed through a detecting system (not shown). Then, this process mayinclude a sorting process by a sorting device or the like, depending onthe individual physical property such as the DNA content of each cellintroduced, through the differential application of charge to thevarious droplets as those skilled in the art readily understand.Subsequently, each cell is sorted depending on its charge. As mentionedearlier, these general aspects of flow cytometry are well known and arediscussed in the references mentioned earlier (hereby incorporated byreference).

[0024] In relation to the handling of the sample for the flowcytometer's functions and the sample's viability, two aspects can beimportant: torsional alignment and the sample's axial motion. Each ofthese is discussed separately, however, it should be understood thatthey are not mutually exclusive and can synergistically have effects.This is especially true as it relates to the sample's viability, thatis, the ability of the sample to perform its functions with efficaciesexpected and not substantially affected by the flow cytometryprocessing. The first of these two aspects, torsional alignment, isdiscussed first.

[0025] The aspects illustrated in FIG. 1 can also be seen through thethree-dimensional view shown in FIG. 2. This three dimensional viewshows a portion of the flow sheath container (2), the sample injectiontube (3) and the nozzle system having a nozzle (6). The sample injectiontube (3), as shown in FIG. 2A in detail, has a beveled tip (4) and acircular mouth (5). The specially designed nozzle (6) is termed a singletorsional orientation nozzle in the present invention and will bepresented in detail below.

[0026] As is known, the sample injection tube serves to introduce thesample material in thin flow into the nozzle system where the sample issurrounded by the sheath fluid. As is well known for those having theordinary skill in the art, the conventional sample injection tube oftenhas a cylindrical shape. However, since this type of sample injectiontube may not help in controlling orientation of the sample, the samplecoming out from this type of the sample injection tube usually has anon-oriented status. In last a couple of decades, a modified sampleinjection tube was produced (Dean et al., 1978, supra; Fulwyler, 1977,supra; Johnson et al., 1986, Cytometry 7: 268-273; Pinkel et al., 1982,supra). This modified sample injection tube may have a beveled tip andcan help, to some degree, in orienting the sample material coming out ofits tip. Because of the beveled shape at the tip of the sample injectiontube, the sample stream can be drawn into a thin ribbon by the sheathfluid. The resulting change in flow condition can cause a correspondingorientation of the sample material.

[0027] In the present design, based upon the concept of the mechanism ofthe beveled tip, the sample injection tube with the type of beveled tipshown is maintained but the specific interior size is unique. Mostimportant, the location of the beveled tip within the nozzle isespecially established. As shown in FIG. 2A, the sample injection tube(3), termed here an orientation-improving sample injection tube,comprises a beveled tip (4) and a circular mouth (5) in its crosssection. The beveled tip is more or less a rectangular shape in itscross section. This has a long axis and a short axis. While naturallythis may be varied to suit the application or the particles beingsorted, in one preferred embodiment the angle of the beveled tip isabout 4°, the outer diameter of the tube is about 1.5 mm and thediameter of the circular mouth is about 0.25 mm.

[0028] By this far, we have only discussed the role a sample injectiontube plays in orienting the sample. It can be understood that, however,as those skilled in the art should understand, the orientation forcesprovided in this manner are very limited. For example, had this featurealone solved the orientation problems, the efforts thereafter on highpercentage orientation would not have been necessary. Instead, as thoseskilled in the art realized, to get a highly oriented sample, especiallywhen the sample contained the flat, non-spherical, or delicate cellssuch as sperm cells for an insemination purpose or the like, anadditional approach was necessary. As the present invention shows, themajority of the orientation forces should come from the interior surfaceof the nozzle. Thus, the nozzle served as a fundamental element for theproduction of functional, and appropriately powerful yet gentleorientation forces.

[0029] With this understanding, it can now be seen how the presentdesign differs in one regard from the prior art. As can be seen fromFIGS. 1 and 2, and as particularly called out in FIGS. 3A, 3B, and 3C,the flow cytometer system comprises the uniquely designed singletorsional orientation nozzle (6). The single torsional orientationnozzle (6) may be made of some selective materials such as a ceramicmaterial and the like. Although the size of the nozzle, e.g., the heightand the diameter, etc., may be varied, it should preferably fit into aconventional flow cytometer and at the same time provide the desiredorientation forces as described in this present invention. Further,although in one preferred embodiment the nozzle is made into a singlepiece, for the purpose of a better illustration, it may be divided intotwo portions, i.e., an upper cylindrical portion (a) and a lower conicalportion (b). In one of the preferred embodiments, the height of theupper cylindrical portion (a) may be about 8 mm and the outer diametermay be about 6 mm. The height of the conical portion (b) may be about4.5 mm and the outer diam at the orifice may be less than about 1 mm.Thus, the total height of the nozzle may be about 12.5 mm. The use of aunitary nozzle also aids in fixing all orientation and axial motionfactors in an optimum arrangement. It can thus increase ease of use,repeatability, and other practical matters as well.

[0030]FIG. 3A is a three-dimensional view and FIG. 3B & 3C are schematiccross sectional views of a single torsional orientation nozzle of thepresent invention. As can be best illustrated in FIGS. 3A and 3B, Thesingle torsional orientation nozzle (6) comprises a nozzle volumeenclosed by an interior surface element. The interior surface element ofthe single torsional orientation nozzle constitutes its interiorgeometry. The interior surface element may comprise a single torsionalinterior surface element having a single torsional interior surface.This single torsional interior surface element has the ability ofgenerating single torsional hydrodynamic forces having a hydrodynamicaxis when a flow containing the sample passes through it. The singletorsional interior surface element also has a velocity acceleratingcharacteristic that may produce an accelerating velocity upon thesample. When the sample passes through this single torsional interiorsurface element, the sample may be oriented by the single torsionalhydrodynamic forces and radially aligned with respect to thehydrodynamic axis. It may also be accelerated to exit for the subsequentanalysis and sorting process. These special single torsionalhydrodynamic forces may be referred to as single torsional orientationforces.

[0031] The overall shape of the single torsional interior surface isgradually tapered downstream so it may be referred as a graduallytapered, single torsional interior surface element. From thelongitudinal section view as shown in FIG. 3B, this gradually tapered,single torsional interior surface element may be viewed in two dimensionas being a “fan-like” shape that opens from the bottom to the top. Thetapered degree of the gradually tapered, single torsional interiorsurface element may vary but may preferably be about 23° from the bottomof the “fan-like” shape to the top so that the desired acceleratingforce may be generated to act upon the sample. In addition, thegradually tapered, single torsional interior surface element may bedivided into a few zones based upon its interior geometry and each zonemay have a laminar flow surface. Basically, the gradually tapered,single torsional interior surface element may be made of a tapered,elliptical-like interior zone (c) having an elliptical-like, singletorsional interior surface and a cylindrical interior zone (d) in thethree-dimensional view. This elliptical-like, single torsional interiorsurface may include different shapes in its cross sections. For example,besides being ellipse-shaped, it may be oval-shaped, or even close to arectangle-shape. Any of these shapes may occur at any location along theelliptical-like, single torsional interior surface just above and belowa demarcation location at which its ellipticity, ovality, or evenrectangularity reaches a maximum or desired degree. As should beunderstood, each of these shapes is intended to be encompassed by theterm “elliptical-like” even though a true mathematical ellipse is notpresent at a given cross section. Similarly, where discussed, the term“circular” need not be perfectly circular—or even circular at all.Again, it may be preferred to be circular, however, other shapes may beequivalent so long as the appropriate function is present.

[0032] Of course, the tapered, elliptical-like interior zone may have amajor axis and a minor axis in its cross sections and the ellipticitymay be smoothly controlled. Thus, depending upon its ellipticityvariation, this tapered, elliptical-like interior zone may be dividedinto the following zones from the top downstream to the bottom:

[0033] 1) an ellipticity-increasing zone (8) with a circular mouth (7)at the top wherein the ratio of the major axis to the minor axis in thecross sections is increasing;

[0034] 2) a desired ellipse demarcation location (9) downstream theellipticity-increasing zone (8) at which the major axis to the minoraxis reaches an optimal ratio that may be a maximum ratio for a sampleas can be best illustrated in FIG. 3A; and

[0035] 3) an ellipticity-decreasing zone (10) wherein the ratio of themajor axis to the minor axis in the cross sections is decreasing.

[0036] Based upon the above described geometry, the two dimensionalshapes of the cross sectional view from the top to the bottom of thetapered, elliptical-like interior zone may undergo transitional changesfrom a circle at the mouth region, to elliptical-like shapes (which mayeven be actual ellipses) with gradually increasing ellipticity (that isthe ratio of major to minor axes—regardless of the shape involved), tothe desired ellipse or the like, to elliptical-like shapes withgradually decreasing ellipticity, and finally to a circle again at theregion where the tapered elliptical-like interior zone joins thecylindrical zone. Since the whole elliptical-like interior zone istapered, the cross sectional areas of the whole elliptical-like interiorzone will become gradually smaller from the top to the bottom. Theellipticity may thus be adjusted by changing the ratio of the major tothe minor axis. The major to the minor axis ratio may gradually changefrom the top from 1 to larger than 1, or perhaps even an optimal ratiofor the sample. The optimal ratio may be a maximum ratio. Subsequently,the ratio may gradually change back from the maximum ratio to smallerthan the maximum ratio and then to 1. As those skilled in the art maywell know, when the ratio becomes 1 the shape in cross section may be acircle. The maximum ratio as referred above may vary to some degree. Ina preferred embodiment, the length of the major axis may be 2.2 mm andthat of the minor axis may be 1.0 mm. Thus, the maximum ratio isdesigned to be about 2.2 for this one preferred embodiment. Naturally itmay vary based on application or the like.

[0037] In one embodiment, the desired ellipse demarcation location (9)downstream the ellipticity-increasing zone (8) within the nozzle may bethe place where the beveled tip of the sample injection tube is located.This may also be the place where the sample in the ribbon flow receivesdesired orientation forces that are fully functional, where the sampleis minimally torqued by the desired orientation forces or torquingforces, where the time required for cell to exit is minimal, or wherethe sample after exiting from the orifice of the nozzle can still wellmaintain its oriented status so the subsequent analysis and sorting canbe conducted efficiently. This location may be referred as an injectionpoint. For the current state-of-the-art high speed sorting flowcytometer now operated, this location or the injection point, based onthe discoveries of the present invention, may be about 6 mm from theexit orifice. Thus, if an orientation maintaining distance is defined asthe distance that indicates how far a sample particle can maintain itsoriented status from the point at which it is oriented to a point atwhich it statistically loses its degree of oriented status, the distancefrom the beveled tip of the sample injection tube to the exit orifice ofthe nozzle and the distance from the exit orifice to the intersectionwith the laser beam or sensor along the flow path in the falling zonewell falls within this orientation maintaining distance. For example, itmay be within 10 mm from the beveled tip to the intersection with thelaser beam, as described by Dean and his colleagues (Dean et al.,supra). Therefore, any sample particles that are oriented, no matter atwhich point within the distance from the beveled tip to the intersectionwith the laser beam or sensor, will maintain their oriented statusbefore they are analyzed. Theoretically, this orientation maintainingdistance could even be longer than 10 mm when a flow cytometer isequipped with the specially designed nozzle of the present invention andthe sample injection tube with the beveled tip. Further, for the fullorientation benefits, the long axis of the beveled tip may be alignedwith the major axis of the desired ellipse demarcation location andshort axis is with the minor axis as shown.

[0038] Downstream from the tapered, elliptical-like interior zone (e)may be a cylindrical interior zone (d). This cylindrical interior zone(d), as can be seen in both FIGS. 3A, 3B, and 3C may be further dividedinto a conical zone (12) that is tapered and a cylindrical zone (14).The conical zone (12) has a larger circular mouth (11) at the top thatjoins with the tapered elliptical-like interior zone (c) and a smallercircular orifice (13) in connection with the cylindrical zone (14). Thelarger circular mouth (11) at the top of the conical zone may be about0.19 mm in diameter and the circular opening may be about 0.07 mm in onepreferred embodiment. The height of the conical zone may be about 0.3mm. The cylindrical zone (14) may also have a mouth with the samediameter as the smaller opening of the conical zone throughout itscircular exit orifice (15) and may be about 0.15 mm in height.

[0039]FIG. 4A illustrates a bottom view of the single torsionalorientation nozzle showing the circular orifice. The circular orificeshould be small enough so that tiny droplets containing sample particlesmay be formed. The diameter in one of the preferred embodiments may beabout 0.07 mm. FIG. 4B shows a top view of the single torsionalorientation nozzle. As can be seen clearly, the mouth may be in acircular shape with a diameter of about 5.25 mm.

[0040] Referring to FIG. 5, it can be seen how orientation occurs. Asmay be noticed, this figure is a modified drawing from Kachel and hiscolleagues (FIG. 3, Kachel et al., 1977, J. Histochem. Cytochem. 25:774-780). This drawing, a cross section around the desired demarcationellipse location (9), shows, first of all, the distributions of theorientation forces generated from the elliptical-like, single torsionalinterior surface. As shown, the dissimilar transformation of theeliptical-like, single torsional interior surface can cause preferentialside forces to generate additional flow components along the major axisand may decrease the forces generated along the minor axis. Thus, theforces generated along the major axis may be viewed as stronger than theforces generated along the minor axis to thus orient a flat particle(16) as shown. The unique design of the present invention shows itssuperiority in that the tapered, elliptical-like interior zone (c) isconnected directly to the cylindrical interior zone (d) and the circularexit orifice (15). This specially designed geometry successfully avoidsthe law of similarity and, therefore, the sample particles that havebeen oriented will be able to individually exit the circular exitorifice and still maintain their orientationally aligned status.

[0041] In addition to the above, the whole tapered, single torsionalinterior surface element may be viewed to comprise a laminar flowsurface. Through a laminar flow and the single torsional orientationforces generated by the laminar flow surface, the sample may be radiallyoriented and aligned along the hydrodynamic axis. The orientationallyaligned sample is thus maintained at the orientationally aligned statuswhen exiting the circular exiting orifice where the sample is split intoindividual particles and the like, is surrounded by a sheath fluid drop,and is analyzed. Therefore, the finally oriented sample can be due tothe combined efforts from the beveled tip of the sample injection tubeand the single torsional orientation interior surface that, because ofthe unique geometry, generates single torsional orientation forces andproduces laminar flow.

[0042] It has to be pointed out that the whole interior surface of thesingle torsional orientation nozzle may be unitary. The way of dividingthe whole interior surface into the tapered, elliptical-like interiorzone (c) the cylindrical interior zone (d) and their own subsequentzones as described above is purely for a clear explanation purpose.

[0043] The animal breeding industry has been increasingly takingadvantage of the principles of flow cytometry and utilizing the benefitsthat a high-speed flow cytometer can provide. Sexed sperm specimens cannow be successfully discriminated by the sorting mechanisms that theflow cytometer employs. With this uniquely designed single torsionalorientation nozzle, the X and Y-chromosome-bearing sperms may be sortedmore efficiently and at a higher percentage as described above. Sexedsperm cells may be buffered in our specially prepared sperm compatiblebuffer as described in PCT Publication No. WO 99/05504 (LoDo PCT). Thebuffered sperm cells may be injected at the demarcation location withinthe elliptical-like, single torsional interior surface element of thesingle torsional orientation nozzle where they may be surrounded by thesheath fluid to form a sheath-surrounded sperm. Subsequently thesperm-containing drops may be produced by a drop-forming mechanism andanalyzed at the free falling area. The sperm-containing drops are thencharged and sorted by the sorting device and colleted by asperm-compatible collecting system containing a specially madesperm-collecting fluid. This whole process may minimize the stressesupon the sperm created through the sorting process. The X orY-chromosome-bearing sperm may then be used for insemination andproduction of a mammal of a desired sex.

[0044] Thus, it is at least the unique design of an interior surfacegeometry of the single torsional orientation nozzle that makes theinvention superior to other conventional nozzle. As will be wellexpected by those having ordinary skill in the art, this singletorsional orientation nozzle, when specially combined with the beveledsample injection tube located at an appropriate location relative to aspecific region of the interior surface of the single torsionalorientation nozzle, can provide results which may be even moresatisfactory.

[0045] As mentioned earlier, the forgoing discussed the torsionalalignment aspect of the invention. A second important aspect is that ofthe sample's axial motion. This aspect encompasses not only the motionof the sample as it traverses the nozzle down the central axis, but thestresses the sample receives during its path. These motions can perhapsbe most easily characterized by three values, the three derivatives ofdistance with respect to location along the sample. These derivativescan be summarized by the following: Analogous more Derivative CommonConcept first derivative of distance with respect to location velocitysecond derivative of distance with respect to location accelerationthird derivative of distance with respect to location rate of change ofacceleration

[0046] As may be understood from FIGS. 6-9 c, the nozzle may present anynumber of axial motion surfaces, that is surfaces which influence orperhaps only confine the sample as it passes through the nozzle. Asshown in FIG. 6, the axial motion surfaces may be symmetric pairs andmay also be as simple as a first axial motion surface (21) and a secondaxial motion surface (22). As the sample passes down the nozzle (6),these axial motion surfaces can act in manners which influence thesample or its viability. The sample is thus (usually hydrodynamically)subjected to a first axial motion surface (21). It may then transitionat a transition location (23) to become influenced by a second axialmotion surface (22). After the transition location (23) the sample isthen subjected to the second axial motion surface (22). It may then exitthe nozzle such as at the circular exit orifice (15).

[0047] It should be understood that the axial motion surfaces can haveany shape. In a system, such as one that may have constant velocity,they may have a tubular shape. As shown in FIGS. 6 & 8, in a system suchas one that achieves acceleration of the sample as it passes through thenozzle (6), they may be configured as acceleration surfaces such as theconical surfaces shown. The acceleration surface could also deceleratethe sample, of course. By causing acceleration or deceleration, thesurface would at so as to change the velocity of the sample as it passesthrough the nozzle (6). Thus, it can be understood that the nozzle(6),such as shown in FIG. 8 may include a first axial acceleration surface(24) and a second axial acceleration surface (25). The first axialacceleration surface (24) causes the sample to experience an a firstacceleration value (which may or may not be constant) and the secondaxial acceleration surface (25) can cause the sample to experience asecond acceleration value. This second acceleration value may or may notbe different from the first. As shown in FIG. 8, since the secondacceleration surface (25) converges at a different rate, it likely wouldindicate a different acceleration value.

[0048] Naturally, anytime there is an acceleration, the sample mayexperience a stress. This stress can have impacts on the samplesviability and functionality. One particular aspect for some samples,such as longer cells, may be the fact that when there is a change invelocity, there may be differences in the velocity tendency from one endof the sample to the next. This may be most easily understood inreference to a sample such as a sperm cell. Viable sperm cells haveheads and tails. When the head is accelerated differentially from theacceleration of the tail, or when the head is moved at a velocitydifferent from that of the tail, a differential may be created from headto tail. This differential may cause stress on the cell. In extremecases, it may even pull the tail from the head. Obviously this coulddestroy the efficacy of the sample. The present invention provides asystem through which this can be minimized and the undesirable effectscan be avoided or reduced. This is accomplished by subjecting the sampleto a “low” degree of changes in acceleration or velocity across thesample's length. As those in the art would be able to understand, “low”may be a relative term which can depend on the cell and the environment.It may be theoretically or empirically determine as a value which isshown to achieve practical percentages of efficacy in the sample for itsspecific application. These probabilities may be such as at least 70%,80%, 90%, or the like. The “low” acceleration or rate of change inacceleration may also be affirmatively applied.

[0049] Changes in acceleration or velocity can occur when the axialmotion surfaces change. These changes can be abrupt or gentle. Naturallysome embodiments of the present invention prefer the latter. Referringto FIG. 6, it can be seen how an abrupt change in the axial motionsurface along the axis can stress the sample. The first axial motionsurface (21) changes in a discrete fashion at the transition location(23). For example, when the second axial motion surface (22) is createdby a separate element, such as by the insertion of a jewel, there canexist a discontinuity in the nozzle (6). At such a point, the sample canthen be subjected to an extreme change in velocity almostinstantaneously. Note that such a discrete change may existunintentionally, due to almost imperceptible misalignments. Regardless,aspects such as these can tend to pull the sample apart. By providingtransitions which may not be discrete the present invention can avoid orminimize the stresses thus created. The transition can be a continuoustransition as in curved area, by having a limited amount of‘discreteness’ or misalignment, or may just avoid the possibility of adiscrete change by having an inner surface on the nozzle (6) which isunitary. In this manner, the nozzle may effectively have a unitarysurface. In such an arrangement, the nozzle (6) can be affirmativelydesigned so as to present a transition with a maximal accelerationdifferentiation. As shown in FIG. 8, this may be done through designingin a limited maximal acceleration differentiation transition area (26)such as shown between the first axial motion surface and the second. Itmay also be accomplished by using a unitary exit orifice. The limitedmaximal acceleration differentiation transition area can then be at oras a result of the unitary exit orifice.

[0050] In terms of the three derivatives of distance with respect tolocation mentioned earlier, the above concepts can be understood byreference to FIGS. 7a-c and 9 a-c. As shown, these figures are graphicalrepresentations of the three derivative values at respective locationsin their adjacent nozzles shown in FIGS. 6 & 8. FIGS. 7a and 9 arepresent the first derivative of distance with respect to location, aconcept similar to velocity. Since the nozzle in FIG. 6 has a discretechange at the transition location (23), it can be seen that dl/dlchanges discretely at the transition location (23). For the nozzle (6)in FIG. 8, the dl/dl value does not discretely change. This the samplemay be treated to less stress for this reasons alone. In FIGS. 7b and 9b, it can be seen that the d²l/dl² values for their respective nozzleare also different. In FIG. 7b, the second derivative of distance withrespect to location value (or perhaps more easily viewed asacceleration) has a moment of extreme change. Again, this is not sopresent in FIG. 9b. Finally, the third derivative of distance withrespect to location values, d ³l/dl³, (or perhaps more easily viewed asrate of change of acceleration) also differ. In FIG. 7c, the value firstgoes positive and then negative. In the values shown in FIG. 9c, thevalues never change signs, they are either zero or positive, but nevernegative. Each of these concepts can be conveniently constructed throughwhich to understand and characterize the nozzle as it is designed toavoid or minimize stresses on the sample.

[0051] An other aspect which may be a factor for some samples is theaspect of the duration of the velocity, acceleration, or rate of changeof acceleration as experienced by the sample. This may also be referredto as the dwell time for the sample. In flow cytometry, there is often aneed for single samples to be placed in single drops. Aspects such asthis can cause a desire to transition the fluid at the last possibletime. In systems which attempt to do this, it can be important to payparticular attention to areas in the vicinity is about 100 um of theexit point (27), areas more than 300 um away from the exit point (27),areas in the vicinity of the exit point (27), or even areas away fromthe exit point (27). In addition, in some systems it may be acceptableto only momentarily subject the sample to the undesired values. Thuslimits can be established throughout the nozzle (6) or at specificlocations within the nozzle (6). Some of the limits which can be appliedare set forth in table s 1 & 2. TABLE 1 d²l/dl² values 0.16 m/sec permicron in the nozzle, 0.05 m/sec per micron in the nozzle, the abovevalues away from the vicinity of the exit point, 0.10 m/sec per micronaway from the exit point, 0.13 m/sec per micron away from the exitpoint, 0.16 m/sec per micron in the vicinity of the exit point, 0.20m/sec per micron in the vicinity of the exit point, 0.23 m/sec permicron in the vicinity of the exit point, 100 × 10⁻³ m/sec per micron ata distance of more than 300 um away from the exit point, 50 × 10⁻³ m/secper micron at a distance of more than 300 um away from the exit point,25 × 10⁻³ m/sec per micron at a distance of more than 300 um away fromthe exit point, values which do not discontinuously change along acentral axis, values which are at most any of the above, any of thesevalues at various locations, any combination of these values, anycombinations of any of these values with any of the values in Table 2.

[0052] TABLE 2 d³l/dl³ values 100,000 × 10⁻⁶ m/sec per micron² in thenozzle, 10,000 × 10⁻⁶ m/sec per micron² in the nozzle, 2,000 × 10⁻⁶m/sec per micron² in the nozzle, 1,100 × 10⁻⁶ m/sec per micron² in thenozzle, the above values away from the vicinity of the exit point,100,000 × 10⁻⁶ m/sec per micron² away from the exit point, 50,000 × 10⁻⁶m/sec per micron² away from the exit point, 10,000 × 10⁻⁶ m/sec permicron² away from the exit point, 5,000 × 10⁻⁶ m/sec per micron² awayfrom the exit point, 1,000 × 10⁻⁶ m/sec per micron² away from the exitpoint, 300 × 10⁻⁶ m/sec per micron² away from the exit point, 200 × 10⁻⁶m/sec per micron² at a distance from the exit point, 100 × 10⁻⁶ m/secper micron² at a distance from the exit point, a rate of change ofacceleration values with respect to axial location as do notdiscontinuously change in the nozzle, a rate of change of accelerationvalues or d³l/dl³ values as do not change sign in the nozzle, valueswhich are at most any of the above, any combination of the above valuesat various locations, any combination of the above. any of these valuesat various locations, any combination of these values, any combinationsof any of these values with any of the values in Table 1

[0053] In affirmatively coordinating such aspects with specific samples,the values may also be established over the effective cell/samplelength. These lengths can be both theoretically determined, measured asthe actual sample length, or even be empirically determined as aneffective sample length. Again, these affirmative or coordinated actionsresult in avoiding leaving things to chance and can permit certainty forusers. In the empirical determinations, among others, it should beunderstood that the values achieved may be chosen so as to not exceedthe practical capabilities of the sample over its length, that is sothat the sample retain a sufficiently acceptable probability offunctionality after they are processed. In these manners, bycoordinating the maximal acceleration differentiation, by affirmativelylimiting the maximal acceleration differentiation, and by affirmativelychoosing values (determined or not) so as to not exceed the practicalcapabilities of the sample, the present invention can achieve its ends.

[0054] As mentioned earlier, synergy can exist between this aspect andthe hydrodynamic alignment aspect of the invention. The combined twistand pull can and apparently does cause stress in some samples,especially sperm cells. Thus the possibility of combining the torsionalhydrodynamic forces and the maximal acceleration differentiation or thelike values, these aspects can combine to minimize stress as well. Therecan also be considered the aspect of combining the above values andconcepts with other parameters which are likely to cause stress in aflow cytometer setting. Such parameters may include operation at sortrates of at least 500 sorts per second, at least 1000 sorts per second,and at least 1500 sorts per second. Similarly, this can also includeoperations at 50 psi and the like. Finally, as alluded to above, certainsamples can particularly susceptible to stress, to the aspects mentionedabove, or to the values set forth above. This can be particularly trueof sperm cells, sperm collection systems, bovine sperm cells, equinesperm cells, sperm cells which have been stained and sorted by their DNAcontent (such as in sexed sperm cells), sorted male or female bovinesperm cells, and even sorted male or female equine sperm cells.

[0055] As can be easily understood from the foregoing, the basicconcepts of the present invention may be embodied in a variety of ways.It involves both exercise techniques as well as devices to accomplishthe appropriate exercise. In this application, the exercise techniquesare disclosed as part of the results shown to be achieved by the variousdevices described and as steps which are inherent to utilization. Theyare simply the natural result of utilizing the devices as intended anddescribed. In addition, while some devices are disclosed, it should beunderstood that these not only accomplish certain methods but also canbe varied in a number of ways. Importantly, as to all of the foregoing,all of these facets should be understood to be encompassed by thisdisclosure.

[0056] The discussion included in this application is intended to serveas a basic description. The reader should be aware that the specificdiscussion may not explicitly describe all embodiments possible; manyalternatives are implicit. It also may not fully explain the genericnature of the invention and may not explicitly show how each feature orelement can actually be representative of a broader function or of agreat variety of alternative or equivalent elements. Again, these areimplicitly included in this disclosure. Where the invention is describedin device-oriented terminology, each element of the device implicitlyperforms a function. Apparatus claims may not only be included for thedevice described, but also method or process claims may be included toaddress the functions the invention and each element performs. Neitherthe description nor the terminology is intended to limit the scope ofthe claims which will be included in a full patent application.

[0057] It should also be understood that a variety of changes may bemade without departing from the essence of the invention. Such changesare also implicitly included in the description. They still fall withinthe scope of this invention. A broad disclosure encompassing both theexplicit embodiment(s) shown, the great variety of implicit alternativeembodiments, and the broad methods or processes and the like areencompassed by this disclosure and may have been relied upon whendrafting the claims for this application. This application will seekexamination of as broad a base of claims as deemed within theapplicant's right and will be designed to yield a patent coveringnumerous aspects of the invention both independently and as an overallsystem.

[0058] Further, each of the various elements of the invention and claimsmay also be achieved in a variety of manners. This disclosure should beunderstood to encompass each such variation, be it a variation of anembodiment of any apparatus embodiment, a method or process embodiment,or even merely a variation of any element of these. Particularly, itshould be understood that as the disclosure relates to elements of theinvention, the words for each element may be expressed by equivalentapparatus terms or method terms—even if only the function or result isthe same. Such equivalent, broader, or even more generic terms should beconsidered to be encompassed in the description of each element oraction. Such terms can be substituted where desired to make explicit theimplicitly broad coverage to which this invention is entitled. As butone example, it should be understood that all actions may be expressedas a means for taking that action or as an element which causes thataction. Similarly, each physical element disclosed should be understoodto encompass a disclosure of the action which that physical elementfacilitates. Regarding this last aspect, as but one example, thedisclosure of an “orientation nozzle” should be understood to encompassdisclosure of an “orienting element”, the act of “orienting”—whetherexplicitly discussed or not—and, conversely, were there only disclosureof the act of “orienting”, such a disclosure should be understood toencompass disclosure of an “orientation element” and even a “means fororienting”. Such changes and alternative terms are to be understood tobe explicitly included in the description.

[0059] All references in the disclosure or listed in the informationdisclosure statement filed with the application are hereby incorporatedby reference; however, to the extent statements might be consideredinconsistent with the patenting of this/these invention(s) suchstatements are expressly not to be considered as made by theapplicant(s).

[0060] Finally, unless the context requires otherwise, it should beunderstood that the term “comprise” or variations such as “comprises” or“comprising”, are intended to imply the inclusion of a stated element orstep or group of elements or steps but not the exclusion of any otherelement or step or group of elements or steps. Such terms should beinterpreted in their most expansive form so as to afford the applicantthe broadest coverage legally permissible in countries such as Australiaand the like.

What is claimed is:
 1. A flow cytometer system, comprising: a. a sampleinjection tube having an injection point through which a sample may beintroduced; b. a sheath fluid container having a bottom end and whereinsaid sample injection tube is located within said sheath fluidcontainer; c. a sheath fluid port connected to said sheath fluidcontainer; d. a single torsional orientation nozzle located at least inpart below said injection point; and e. an analytical system whichsenses below said single torsional orientation nozzle.
 2. A flowcytometer system as described in claim 1 wherein said single torsionalorientation nozzle comprises a single torsional interior surfaceelement.
 3. A flow cytometer system as described in claim 2 wherein saidsingle torsional interior surface element comprises a tapered,elliptical-like, single torsional interior surface element.
 4. A flowcytometer system as described in claim 1 and further comprising: a. afirst axial motion surface in said nozzle; b. a second axial motionsurface in said nozzle; and c. a limited maximal accelerationdifferentiation transition area between said first axial motion surfacein said nozzle and said second axial motion surface in said nozzlewherein said limited maximal acceleration differentiation transitionarea is coordinated with said sample so as to be affirmatively limitedto not exceed the practical capabilities of said sample over its length.5. A flow cytometer system as described in claim 4 wherein said firstaxial motion surface comprises a first axial acceleration surface andwherein said second axial motion surface comprises a second axialacceleration surface.
 6. A flow cytometer system as described in claim 5wherein said nozzle has acceleration values caused by its internalsurface and wherein said acceleration values are selected from a groupcomprising: not more than about 0.16 m/sec per micron, not more thanabout 0.05 m/sec per micron away from the vicinity of the exit orifice,not more than about 0.10 m/sec per micron away from the vicinity of theexit orifice, not more than about 0.13 m/sec per micron away from thevicinity of the exit orifice, not more than about 0.16 m/sec per micronin the vicinity of the exit orifice, not more than about 0.20 m/sec permicron in the vicinity of the exit orifice, not more than about 0.23m/sec per micron in the vicinity of the exit orifice, not more thanabout 100×10⁻³ m/sec per micron at a distance of more than 300 um awayfrom the exit orifice, not more than about 50×10⁻³ m/see per micron at adistance of more than 300 um away from the exit orifice, not more thanabout 25×10⁻³ m/sec per micron at a distance of more than 300 um awayfrom the exit orifice, such acceleration values with respect to axiallocation as do not discontinuously change along a central axis, not morethan about 100,000×10⁻⁶ m/sec per micron², not more than about10,000×10⁻⁶ m/sec per micron², not more than about 2,000×10⁻⁶ m/sec permicron², not more than about 1,100×10⁻⁶ m/sec per micron², not more thanabout 100,000×10⁻⁶ m/sec per micron² away from the vicinity of the exitorifice, not more than about 50,000×10⁻⁶ m/sec per micron² away from thevicinity of the exit orifice, not more than about 10,000×10⁻⁶ m/sec permicron² away from the vicinity of the exit orifice, not more than about5,000×10⁻⁶ m/sec per micron² away from the vicinity of the exit orifice,not more than about 1,000×10⁻⁶ m/sec per micron² away from the vicinityof the exit orifice, not more than about 300×10⁻⁶ m/sec per micron² awayfrom the vicinity of the exit orifice, not more than about 200×10⁻⁶m/sec per micron² at a distance of more than 300 um away from the exitorifice, not more than about 100×10⁻⁶ m/sec per micron² at a distance ofmore than 300 um away from the exit orifice, such rate of change ofacceleration values with respect to axial location as do notdiscontinuously change along a central axis, and such rate of change ofacceleration values with respect to axial location as do not change signalong a central axis away from the vicinity of the exit orifice.
 7. Aflow cytometer system as described in claim 4 wherein said limitedmaximal acceleration differentiation transition area comprises a unitarysurface.
 8. A flow cytometer system as described in claim 4 wherein saidlimited maximal acceleration differentiation transition area comprises aunitary exit orifice.
 9. A flow cytometer system as described in claim 4wherein said analytical system which senses below said nozzle operatesat a rate selected from a group comprising at least 500 sorts persecond, at least 1000 sorts per second, and at least 1500 sorts persecond.
 10. A flow cytometer system as described in claim 4 and furthercomprising a pressurization system which operates at least about 50 psi.11. A flow cytometer system as described in claim 9 and furthercomprising a sperm collection system.
 12. A flow cytometer system asdescribed in claim 10 and further comprising a sperm collection system.13. A flow cytometer system as described in claims 4, 7, 8, 9, or 10wherein said sample comprises sperm cells selected from a groupcomprising bovine sperm cells and equine sperm cells.
 14. A sexed spermspecimen produced with a flow cytometer system as described in claims 11or
 12. 15. A flow cytometer system as described in claim 14 wherein saidsample comprises sperm cells selected from a group comprising bovinesperm cells and equine sperm cells.
 16. A mammal produced through use ofa sexed sperm specimen produced with a flow cytometer system asdescribed in claims 11 or
 12. 17. A flow cytometer system as describedin claim 16 wherein said sample comprises sperm cells selected from agroup comprising bovine sperm cells and equine sperm cells.
 18. A flowcytometer system as described in claim 3 wherein said tapered,elliptical-like, single torsional interior surface element comprises: a.an ellipse-like demarcation location located at about said injectionpoint; and b. an ellipticity-decreasing zone extending from below saidellipse-like demarcation location.
 19. A flow cytometer system asdescribed in claim 3 wherein said tapered, elliptical-like, singletorsional interior surface element comprises: a. anellipticity-increasing zone; b. an ellipse-like demarcation locationdownstream from said ellipticity-increasing zone; and c. anellipticity-decreasing zone extending from said ellipse-like demarcationlocation.
 20. A flow cytometer system as described in claim 18 andfurther comprising: a. a conical zone located below saidellipticity-decreasing zone; b. a cylindrical zone located below saidconical zone; wherein both said conical zone and said cylindrical zonecomprises a laminar flow surface; c. a circular exit orifice locatedbelow said cylindrical zone; d. an oscillator to which said circularexit orifice is responsive; and e. a flow cytometry sorting system belowsaid single torsional orientation nozzle.
 21. A flow cytometer system asdescribed in claim 19 and further comprising: a. a conical zone locatedbelow said ellipticity-decreasing zone; b. a cylindrical zone locatedbelow said conical zone; wherein both said conical zone and saidcylindrical zone comprises a laminar flow surface; and c. a circularexit orifice located below said cylindrical zone; d. an oscillator towhich said circular exit orifice is responsive; and e. a flow cytometrysorting system below said single torsional orientation nozzle.
 22. Aflow cytometer system as described in claims 18 wherein said tapered,elliptical-like, single torsional interior surface element, said conicalzone, and said cylindrical zone are unitary.
 23. A flow cytometer systemas described in claims 20 wherein said tapered, elliptical-like, asingle torsional interior surface element, said conical zone, saidcylindrical zone, and said circular exit orifice are unitary.
 24. A flowcytometer system as described in claim 22 wherein said ellipse-likedemarcation location has a major axis and a minor axis having a ratio,and wherein said ratio of said major axis to minor axis comprises anoptimal ratio for a sample.
 25. A flow cytometer system as described inclaim 24 wherein said major axis of said desired ellipse-likedemarcation location is about 2.2 mm and wherein said minor axis of saiddesired ellipse-like demarcation location is about 1.0 mm.
 26. A flowcytometer system as described in claim 19 wherein said ellipse-likedemarcation location has a major axis and a minor axis, wherein saidmajor axis of said desired ellipse-like demarcation location is about2.2 mm and wherein said minor axis of said desired ellipse-likedemarcation location is about 1.0 mm.
 27. A flow cytometer system asdescribed in claim 22 wherein said single torsional orientation nozzlehas a downstream direction, wherein said ellipticity-decreasing zone hascross sections and cross section areas, wherein said cross sections ofsaid ellipticity-decreasing zone undergo transitional changes fromellipse-like shapes to circular shapes downstream, and wherein saidcross section areas become progressively smaller downsteam.
 28. A flowcytometer system as described in claim 27 wherein each of said crosssections of said ellipticity-decreasing zone has a major axis and aminor axis and wherein said major axis and said minor axis progressivelybecome equal downstream.
 29. A flow cytometer system as described inclaim 21 wherein said conical zone is about 0.3 mm in height.
 30. A flowcytometer system as described in claim 29 wherein said cylindrical zoneis about 0.15 mm in height.
 31. A flow cytometer system as described inclaim 2 wherein said single torsional interior surface element comprisesa gradually tapered, single torsional interior surface element.
 32. Aflow cytometer system as described in claim 31 wherein said graduallytapered, single torsional interior surface element comprises an interiorsurface element that tapers at about 23°.
 33. A flow cytometer system asdescribed in claim 19 wherein said tapered, single torsional interiorsurface element comprises an interior surface element that tapers atabout 23°.
 34. A flow cytometer system as described in claim 23 whereinsaid single torsional orientation nozzle comprises a single torsional,ceramic orientation nozzle.
 35. A flow cytometer system as described inclaim 22 wherein said single torsional orientation nozzle has a heightand a top with an outer diameter, and wherein said height is about 13mm, and wherein said outer diameter about 6 mm.
 36. A flow cytometersystem as described in claim 20 wherein said flow cytometer systemcomprises a circular exit orifice, and wherein said tapered,elliptical-like, single torsional interior surface element has a mouthand wherein said mouth is about 5.25 mm in diameter and said circularexit orifice is about 0.07 mm in diameter.
 37. A flow cytometer systemas described in claim 35 wherein said flow cytometer system comprises acircular exit orifice, and wherein said tapered, elliptical-like, singletorsional interior surface element has a mouth and wherein said mouth isabout 5.25 mm in diameter and said circular exit orifice is about 0.07mm in diameter.
 38. A flow cytometer system as described in claim 36wherein said conical zone has a top with an inner diameter, and whereinsaid inner diameter at said top of said conical zone is about 0.19 mm.39. A flow cytometer system as described in claim 37 wherein saidconical zone has a top with an inner diameter, and wherein said innerdiameter at said top of said conical zone is about 0.19 mm.
 40. A flowcytometer system as described in claim 18 wherein said sample injectiontube comprises an orientation-improving sample injection tube.
 41. Aflow cytometer system as described in claim 40 wherein saidorientation-improving sample injection tube comprises a beveled tip. 42.A flow cytometer system as described in claim 41 wherein said beveledtip has a circular mouth and wherein said circular mouth has a diameterof about 0.01 mm.
 43. A flow cytometer system as described in claim 41wherein said tapered, elliptical-like interior zone has a major axis anda minor axis at said injection point, and wherein said major axis ofsaid beveled tip is aligned with said major axis of said tapered,elliptical-like interior zone at said injection point.
 44. A flowcytometer system as described in claim 43 wherein said single torsionalorientation nozzle has a bottom, wherein said beveled tip has a circularmouth, wherein said flow cytometer system further comprises a circularexit orifice located at said bottom of said single torsional orientationnozzle, and wherein said injection point is located at a distance fromsaid circular exit orifice of said single torsional orientation nozzleat which a sample exiting from said circular mouth of said beveled tipreceives minimal torquing forces to achieve an orientationally alignedstatus.
 45. A flow cytometer system as described in claim 44 whereinsaid injection point is located at a distance from said circular exitorifice at which said orientationally aligned status of said sample issubstantially maintained when said sample exits said circular orifice ofsaid single torsional orientation nozzle.
 46. A flow cytometer system asdescribed in claim 45 wherein said injection point is located about 6 mmfrom said circular exit orifice of said single torsional orientationnozzle.
 47. A flow cytometer system as described in claim 9 wherein saidflow cytometer system has dimensions established according to claims 26,29, 30, 36, 38 or
 46. 48. A flow cytometer system as described in claim1 wherein said sample comprises sperm cells in a sperm compatiblebuffer.
 49. A flow cytometer system as described in claim 48 whereinsaid analytical system comprises a flow cytometry sorting system.
 50. Aflow cytometer system as described in claim 49 and further comprising asperm compatible collection system.
 51. A flow cytometer system asdescribed in claim 49 wherein said sample comprises sperm cells in asperm compatible buffer and wherein said sperm cells are selected from agroup consisting of equine sperm cells and bovine sperm cells.
 52. Aflow cytometer system as described in claim 51 wherein said flowcytometer system has dimensions established according to claims 26, 29,30, 36, 38, or
 46. 53. A sexed sperm specimen produced with a flowcytometer system as described in any of claims 1, 20, 23, 24, 26, 29,30, 32, 39, 41, 44, 45, 51, or
 52. 54. A mammal produced through use ofa sexed sperm specimen produced with a flow cytometer system asdescribed in any of claims 1, 20, 23, 24, 26, 29, 30, 32, 39, 41, 44,45, 51, or
 52. 55. A flow cytometer system as described in claims 18,23, 26, 29, 30, 32, 37, 39, 42, 46 and further comprising: a. a firstaxial motion surface in said nozzle; b. a second axial motion surface insaid nozzle; and c. a limited maximal acceleration differentiationtransition area between said first axial motion surface in said nozzleand said second axial motion surface in said nozzle wherein said limitedmaximal acceleration differentiation transition area is coordinated withsaid sample so as to be affirmatively limited to not exceed thepractical capabilities of said sample over its length.
 56. A flowcytometer system as described in claim 55 wherein said limited maximalacceleration differentiation transition area comprises a unitarysurface.
 57. A flow cytometer system as described in claim 55 whereinsaid limited maximal acceleration differentiation transition areacomprises a unitary exit orifice.
 58. A flow cytometer system asdescribed in claim 55 wherein said analytical system which senses belowsaid nozzle operates at a rate selected from a group comprising at least500 sorts per second, at least 1000 sorts per second, and at least 1500sorts per second.
 59. A flow cytometer system as described in claim 55and further comprising a pressurization system which operates at leastabout 50 psi.
 60. A flow cytometer system as described in claim 58 andfurther comprising a sperm collection system.
 61. A flow cytometersystem as described in claim 59 and further comprising a spermcollection system.
 62. A flow cytometer system as described in claim 55wherein a said sample comprises sperm cells selected from a groupcomprising bovine sperm cells and equine sperm cells.
 63. A flowcytometer system as described in claim 57 wherein a said samplecomprises sperm cells selected from a group comprising bovine spermcells and equine sperm cells.
 64. A flow cytometer system as describedin claim 58 wherein a said sample comprises sperm cells selected from agroup comprising bovine sperm cells and equine sperm cells.
 65. A flowcytometer system as described in claim 59 wherein a said samplecomprises sperm cells selected from a group comprising bovine spermcells and equine sperm cells.
 66. A sexed sperm specimen produced with aflow cytometer system as described in claim
 60. 67. A sexed spermspecimen produced with a flow cytometer system as described in claim 64.68. A mammal produced through use of a sexed sperm specimen producedwith a flow cytometer system as described in claim
 60. 69. A mammalproduced through use of a sexed sperm specimen produced with a flowcytometer system as described in claim
 64. 70. A method of flowcytometry sample processing, comprising the steps of: a. establishing asheath fluid; b. injecting a sample into said sheath fluid at aninjection point; c. establishing a single torsional surface in a nozzlehaving a central axis around which a torque is applied; d. generatingsingle torsional hydrodynamic forces from said single torsional surface;e. orienting said sample with said single torsional hydrodynamic forces;f. exiting said sample from said nozzle; g. analyzing said sample.
 71. Amethod of flow cytometry sample processing as described in claim 70wherein said step of establishing a single torsional surface comprisesthe step of utilizing a single torsional interior surface in saidnozzle.
 72. A method of flow cytometry sample processing as described inclaim 71 wherein said step of establishing a single torsional surface ina nozzle comprises the step of establishing a tapered, elliptical-like,single torsional interior surface in said nozzle.
 73. A method of flowcytometry sample processing as described in claim 72 wherein saidtapered elliptical-like, single torsional interior surface has anellipticity which varies along its length and further comprising thestep of smoothly varying said ellipticity of said elliptical-like,single torsional interior surface.
 74. A method of flow cytometry sampleprocessing as described in claim 70 and further comprising the steps of:a. subjecting said sample to a first axial motion surface in a nozzle;b. transitioning to a second axial motion surface in said nozzle; c.subjecting said sample to said second axial motion surface in saidnozzle wherein said first and said second axial motion surfacestransition with a maximal acceleration differentiation; d. coordinatingsaid maximal acceleration differentiation so as to not exceed thepractical capabilities of said sample over its length; and e.affirmatively limiting said maximal acceleration differentiation so asto not exceed the practical capabilities of said sample over its length.75. A method of flow cytometry sample processing as described in claim74 wherein said step of subjecting said sample to a first axial motionsurface in a nozzle comprises the step of subjecting said sample to afirst axial acceleration surface in said nozzle and wherein said step ofsubjecting said sample to said second axial motion surface in saidnozzle comprises the step of subjecting said sample to a second axialacceleration surface wherein said first and said second axial motionsurfaces transition with a maximal acceleration differentiation.
 76. Amethod of flow cytometry sample processing as described in claim 75wherein said nozzle creates acceleration values though its internalsurface and wherein said acceleration values are selected from a groupcomprising: not more than about 0.16 m/sec per micron, not more thanabout 0.05 m/sec per micron away from the vicinity of the exit orifice,not more than about 0.10 m/sec per micron away from the vicinity of theexit orifice, not more than about 0.13 m/sec per micron away from thevicinity of the exit orifice, not more than about 0.16 m/sec per micronin the vicinity of the exit orifice, not more than about 0.20 m/sec permicron in the vicinity of the exit orifice, not more than about 0.23m/sec per micron in the vicinity of the exit orifice, not more thanabout 100×10⁻³ m/sec per micron at a distance of more than 300 um awayfrom the exit orifice, not more than about 50×10⁻³ m/sec per micron at adistance of more than 300 um away from the exit orifice, not more thanabout 25×10⁻³ m/sec per micron at a distance of more than 300 um awayfrom the exit orifice, such acceleration values with respect to axiallocation as do not discontinuously change along a central axis, not morethan about 100,000×10⁻⁶ m/sec per micron², not more than about10,000×10⁻⁶ m/sec per micron², not more than about 2,000×10⁻⁶ m/sec permicron², not more than about 1,100×10⁻⁶ m/sec per micron², not more thanabout 100,000×10⁻⁶ m/sec per micron² away from the vicinity of the exitorifice, not more than about 50,000×10⁻⁶ m/sec per micron² away from thevicinity of the exit orifice, not more than about 10,000×10⁻⁶ m/sec permicron² away from the vicinity of the exit orifice, not more than about5,000×10⁻⁶ m/sec per micron² away from the vicinity of the exit orifice,not more than about 1,000×10⁻⁶ m/sec per micron² away from the vicinityof the exit orifice, not more than about 300×10⁻⁶ m/sec per micron² awayfrom the vicinity of the exit orifice, not more than about 200×10⁻⁶m/sec per micron² at a distance of more than 300 um away from the exitorifice, not more than about 100×10⁻⁶ m/sec per micron² at a distance ofmore than 300 um away from the exit orifice, such rate of change ofacceleration values with respect to axial location as do notdiscontinuously change along a central axis, and such rate of change ofacceleration values with respect to axial location as do not changesigns along a central axis away from the vicinity of the exit orifice.77. A method of flow cytometry sample processing as described in claim74 wherein said single torsional hydrodynamic forces and said maximalacceleration differentiation combine and are affirmatively chosen so asto not exceed the practical capabilities of said sample over its length.78. A method of flow cytometry sample processing as described in claim77 wherein said step of transitioning to a second axial motion surfacein said nozzle comprises the step of subjecting said sample to a unitarysurface.
 79. A method of flow cytometry sample processing as describedin claim 78 wherein said step of transitioning to a second axial motionsurface in said nozzle comprises the step of subjecting said sample to aunitary exit orifice.
 80. A method of flow cytometry sample processingas, described in claim 74 and further comprising the steps of: a.forming drops around said sample after it has exited said nozzle; and b.sorting said drops at a rate selected from the group comprising at least500 sorts per second, at least 1000 sorts per second, and at least 1500sorts per second.
 81. A method of flow cytometry sample processing asdescribed in claim 74 and further comprising the step of pressurizingsaid nozzle at a pressure of at least 50 psi.
 82. A method of flowcytometry sample processing as described in claim 80 wherein said stepof injecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 83.A method of flow cytometry sample processing as described in claim 81wherein said step of injecting a sample into said sheath fluid at aninjection point comprises the step of injecting sperm cells into saidsheath fluid.
 84. A method of flow cytometry sample processing asdescribed in claims 74, 78, 79, 80, or 81 wherein said step of injectinga sample into said sheath fluid at an injection point comprises the stepof injecting sperm cells selected from the group comprising bovine spermcells and equine sperm cells into said sheath fluid.
 85. A method ofcreating a sexed sperm specimen comprising the step of producing a sexedsperm specimen as described in claims 82 or 83 and wherein said step ofinjecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 86.A method of creating a sexed sperm specimen comprising the step ofproducing a sexed sperm specimen as described in claim 85 wherein saidstep of injecting sperm cells into said sheath fluid comprises the stepof injecting sperm cells selected from the group comprising bovine spermcells and equine sperm cells into said sheath fluid.
 87. A method ofcreating a mammal comprising the step of producing a sexed spermspecimen as described in claims 82 or 83 and wherein said step ofinjecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 88.A method of creating a mammal comprising the step of producing a sexedsperm specimen as described in claim 87 wherein said step of injectingsperm cells into said sheath fluid comprises the step of injecting spermcells selected from the group comprising bovine sperm cells and equinesperm cells into said sheath fluid.
 89. A method of flow cytometrysample processing as described in claim 73 wherein said step of smoothlyvarying said ellipticity of said elliptical-like, single torsionalinterior surface comprises the step of decreasing the ellipticity ofsaid elliptical-like, single torsional interior surface downstream fromsaid injection point.
 90. A method of flow cytometry sample processingas described in claim 73 wherein said step of smoothly varying saidellipticity of said elliptical-like, single torsional interior surfacecomprises the steps of: a. increasing the ellipticity of saidelliptical-like, single torsional interior surface downstream in saidnozzle; b. reaching an ellipse-like demarcation location; and c.decreasing the ellipticity of said elliptical-like, single torsionalinterior surface downstream from said ellipse-like demarcation location.91. A method of flow cytometry sample processing as described in claim89 and further comprising the steps of: a. laminarly flowing said sheathfluid within said nozzle; b. subjecting said sheath fluid to a conicalzone; c. subjecting said sheath fluid to a cylindrical zone; d. creatingan exit stream having a circular cross section; e. forming drops fromsaid exit stream; and f. sorting said drops.
 92. A method of flowcytometry sample processing as described in claim 90 and furthercomprising the steps of: a. laminarly flowing said sheath fluid withinsaid nozzle; b. subjecting said sheath fluid to a conical zone; c.subjecting said sheath fluid to a cylindrical zone; d. creating an exitstream having a circular cross section; e. forming drops from said exitstream; and f. sorting said drops.
 93. A method of flow cytometry sampleprocessing as described in claim 91 wherein said step of subjecting saidsheath fluid to a conical zone and said step of subjecting said sheathfluid to a cylindrical zone both comprise the step of utilizing aunitary surface.
 94. A method of flow cytometry sample processing asdescribed in claim 91 wherein said step of subjecting said sheath fluidto a conical zone, and said step of subjecting said sheath fluid to acylindrical zone, and said step of creating an exit stream having acircular cross section all comprise the step of utilizing a unitarysurface.
 95. A method of flow cytometry sample processing as describedin claim 89 wherein said ellipticity has a ratio of a major axis to aminor axis at said injection point, and further comprising the step ofoptimizing said ratio for said sample.
 96. A method of flow cytometrysample processing as described in claim 95 wherein said step ofoptimizing said ratio for said sample comprises the step of setting saidratio at 2.2.
 97. A method of flow cytometry sample processing asdescribed in claim 90 wherein said ellipticity has a ratio of a majoraxis to a minor axis at said injection point, and further comprising thestep of setting said ratio at 2.2.
 98. A method of flow cytometry sampleprocessing as described in claim 93 wherein said elliptical-like, singletorsional interior surface has cross section areas, and wherein saidstep of smoothly varying said ellipticity of said elliptical-like,single torsional interior surface further comprises the step ofdecreasing the cross section areas downstream from said injection point.99. A method of flow cytometry sample processing as described in claim98 wherein said ellipticity has a major and a minor axis and whereinsaid step of smoothly varying said ellipticity of said elliptical-like,single torsional interior surface comprises the step of making saidmajor and a minor axis progressively become equal downstream.
 100. Amethod of flow cytometry sample processing as described in claim 92wherein said step of subjecting said sheath fluid to a conical zonecomprises the step of subjecting said sheath fluid to a conical zone foran optimal length for said sample as it travels downstream.
 101. Amethod of flow cytometry sample processing as described in claim 100wherein said step of subjecting said sheath fluid to a conical zone foran optimal length for said sample as it travels downstream comprises thestep of subjecting said sheath fluid to a 0.3 mm long conical zone. 102.A method of flow cytometry sample processing as described in claim 100wherein said step of subjecting said sheath fluid to a cylindrical zonecomprises the step of subjecting said sheath fluid to a cylindrical zonefor an optimal length for said sample as it travels downstream.
 103. Amethod of flow cytometry sample processing as described in claim 102wherein said step of subjecting said sheath fluid to a cylindrical zonefor an optimal length for said sample as it travels downstream comprisesthe step of subjecting said sheath fluid to a 0.15 mm long cylindricalzone.
 104. A method of flow cytometry sample processing as described inclaim 72 wherein said step of establishing a tapered, elliptical-like,single torsional interior surface in said nozzle comprises the step ofgradually tapering said elliptical-like, single torsional interiorsurface.
 105. A method of flow cytometry sample processing as describedin claim 104 wherein said step of gradually tapering saidelliptical-like, single torsional interior surface comprises the step ofsetting a taper at about 23°.
 106. A method of flow cytometry sampleprocessing as described in claim 90 wherein said step of increasing theellipticity of said elliptical-like, single torsional interior surfacedownstream in said nozzle and decreasing the ellipticity of saidelliptical-like, single torsional interior surface each comprises thestep of setting a taper at about 23°.
 107. A method of flow cytometrysample processing as described in claim 107 wherein said step ofutilizing a unitary surface comprises the step of utilizing a unitaryceramic surface.
 108. A method of flow cytometry sample processing asdescribed in claim 93 wherein said step of utilizing a unitary surfacecomprises the step of establishing a nozzle having a height of about 13mm and an outer diameter of about 6 mm.
 109. A method of flow cytometrysample processing as described in claim 92 wherein said step of creatingan exit stream having a circular cross section comprises the step ofcreating an exit stream having a diameter of about 0.07 mm, and whereinsaid step of smoothly varying said ellipticity of said elliptical-like,single torsional interior surface comprises the step of establishing amouth of about 5.25 mm in diameter.
 110. A method of flow cytometrysample processing as described in claim 108 wherein said step ofcreating an exit stream having a circular cross section comprises thestep of creating an exit stream having a diameter of about 0.07 mm, andwherein said step of smoothly varying said ellipticity of saidelliptical-like, single torsional interior surface comprises the step ofestablishing a mouth of about 5.25 mm in diameter.
 111. A method of flowcytometry sample processing as described in claim 108 wherein said stepof subjecting said sheath fluid to a conical zone comprises the step ofsubjecting said sheath fluid to a conical zone having an inner diameterat a top of said conical zone of about 0.19 mm.
 112. A method of flowcytometry sample processing as described in claim 110 wherein said stepof subjecting said sheath fluid to a conical zone comprises the step ofsubjecting said sheath fluid to a conical zone having an inner diameterat a top of said conical zone of about 0.19 mm.
 113. A method of flowcytometry sample processing as described in claim 89 wherein said stepof injecting a sample into said sheath fluid at an injection pointcomprises the step of assisting in orienting said sample at saidinjection point.
 114. A method of flow cytometry sample processing asdescribed in claim 113 wherein said step of assisting in orienting saidsample at said injection point comprises the step of creating a beveledflow near said injection point.
 115. A method of flow cytometry sampleprocessing as described in claim 114 wherein said step of injecting asample into said sheath fluid at an injection point comprises the stepof establishing a beveled tip having circular mouth with a diameter ofabout 0.01 mm.
 116. A method of flow cytometry sample processing asdescribed in claim 114 and further comprising the step of aligning saidbeveled flow with said tapered, elliptical-like, single torsionalinterior surface in said nozzle.
 117. A method of flow cytometry sampleprocessing as described in claim 70 wherein said step of orienting saidsample with said single torsional hydrodynamic forces comprises the stepof minimally torquing said sample.
 118. A method of flow cytometrysample processing as described in claim 117 wherein said sample travelsa distance after accomplishing said step of generating single torsionalhydrodynamic forces from said single torsional surface and beforeaccomplishing said step of exiting said sample from said nozzle andfurther comprising the step of minimizing said distance.
 119. A methodof flow cytometry sample processing as described in claim 118 whereinsaid step of exiting said sample from said nozzle occurs at an exitorifice and wherein said step of minimizing said distance comprisessetting the distance from said injection point to said exit orifice atabout 6 mm.
 120. A method of flow cytometry sample processing asdescribed in claim 80 wherein said sample is oriented as described inany of claims 97, 101, 103, 109, 111, or
 119. 121. A method of flowcytometry sample processing as described in claim 70 wherein said stepof injecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells in a sperm compatible bufferinto said sheath fluid.
 122. A method of flow cytometry sampleprocessing as described in claim 121 and further comprising the stepsof: a. forming drops around said sperm cells after they have exited saidnozzle; and b. sorting said drops.
 123. A method of flow cytometrysample processing as described in claim 121 and further comprising thestep of collecting said sperm cells after accomplishing said step ofsorting said drops.
 124. A method of flow cytometry sample processing asdescribed in claim 122 wherein said step of injecting sperm cells in asperm compatible buffer into said sheath fluid comprises the step ofinjecting sperm cells in a sperm compatible buffer into said sheathfluid selected from a group consisting of equine sperm cells and bovinesperm cells.
 125. A method of flow cytometry sample processing asdescribed in claim 124 wherein said sample is oriented as described inany of claims 97, 101, 103, 109, 111, or
 119. 126. A method of creatinga sexed sperm specimen comprising the step of producing a sexed spermspecimen as described in any of claims 70, 91, 94, 95, 97, 101, 103,105, 112, 114, 117, 118, 124, or
 125. 127. A method of creating a mammalcomprising the step of producing a sexed sperm specimen as described inany of claims 70, 91, 94, 95, 97, 101, 103, 105, 112, 114, 117, 118,124, or
 125. 128. A method of flow cytometry sample processing asdescribed in claims 89, 94, 97, 101, 103, 105, 110, 112, 115, or 119 andfurther comprising the steps of: a. subjecting said sample to a firstaxial motion surface in a nozzle; b. transitioning to a second axialmotion surface in said nozzle; c. subjecting said sample to said secondaxial motion surface in said nozzle wherein said first and said secondaxial motion surfaces transition with a maximal accelerationdifferentiation; d. coordinating said maximal accelerationdifferentiation so as to not exceed the practical capabilities of saidsample over its length; and e. affirmatively limiting said maximalacceleration differentiation so as to not exceed the practicalcapabilities of said sample over its length.
 129. A method of flowcytometry sample processing as described in claim 128 wherein saidsingle torsional hydrodynamic forces and said maximal accelerationdifferentiation combine and are affirmatively chosen so as to not exceedthe practical capabilities of said sample over its length.
 130. A methodof flow cytometry sample processing as described in claim 129 whereinsaid step of transitioning to a second axial motion surface in saidnozzle comprises the step of subjecting said sample to a unitarysurface.
 131. A method of flow cytometry sample processing as describedin claim 130 wherein said step of transitioning to a second axial motionsurface in said nozzle comprises the step of subjecting said sample to aunitary exit orifice.
 132. A method of flow cytometry sample processingas described in claim 128 and further comprising the steps of: a.forming drops around said sample after it has exited said nozzle; and b.sorting said drops at a rate selected from the group comprising at least500 sorts per second, at least 1000 sorts per second, and at least 1500sorts per second.
 133. A method of flow cytometry sample processing asdescribed in claim 128 and further comprising the step of pressurizingsaid nozzle at a pressure of at least 50 psi.
 134. A method of flowcytometry sample processing as described in claim 132 wherein said stepof injecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 135.A method of flow cytometry sample processing as described in claim 133wherein said step of injecting a sample into said sheath fluid at aninjection point comprises the step of injecting sperm cells into saidsheath fluid.
 136. A method of flow cytometry sample processing asdescribed in claim 128 wherein said step of injecting a sample into saidsheath fluid at an injection point comprises the step of injecting spermcells selected from the group comprising bovine sperm cells and equinesperm cells into said sheath fluid.
 137. A method of flow cytometrysample processing as described in claim 131 wherein said step ofinjecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells selected from the groupcomprising bovine sperm cells and equine sperm cells into said sheathfluid.
 138. A method of flow cytometry sample processing as described inclaim 132 wherein said step of injecting a sample into said sheath fluidat an injection point comprises the step of injecting sperm cellsselected from the group comprising bovine sperm cells and equine spermcells into said sheath fluid.
 139. A method of flow cytometry sampleprocessing as described in claim 133 wherein said step of injecting asample into said sheath fluid at an injection point comprises the stepof injecting sperm cells selected from the group comprising bovine spermcells and equine sperm cells into said sheath fluid.
 140. A method ofcreating a sexed sperm specimen comprising the step of producing a sexedsperm specimen as described in claim 134 and wherein said step ofinjecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 141.A method of creating a sexed sperm specimen comprising the step ofproducing a sexed sperm specimen as described in claim 138 and whereinsaid step of injecting sperm cells into said sheath fluid comprises thestep of injecting sperm cells selected from the group comprising bovinesperm cells and equine sperm cells into said sheath fluid.
 142. A methodof creating a mammal comprising the step of producing a sexed spermspecimen as described in claim 134 and wherein said step of injecting asample into said sheath fluid at an injection point comprises the stepof injecting sperm cells into said sheath fluid.
 143. A method ofcreating a mammal comprising the step of producing a sexed spermspecimen as described in claim 138 and wherein said step of injectingsperm cells into said sheath fluid comprises the step of injecting spermcells selected from the group comprising bovine sperm cells and equinesperm cells into said sheath fluid.
 144. A flow cytometer system,comprising: a. a sample injection tube having an injection point throughwhich a sample may be introduced; b. a sheath fluid container having abottom end and wherein said sample injection tube is located within saidsheath fluid container; c. a sheath fluid port connected to said sheathfluid container; d. a first axial motion surface in a nozzle; e. asecond axial motion surface in said nozzle; f. a limited maximalacceleration differentiation transition area between said first axialmotion surface in said nozzle and said second axial motion surface insaid nozzle wherein said limited maximal acceleration differentiationtransition area is coordinated with said sample so as to beaffirmatively limited to not exceed the practical capabilities of saidsample over its length; and g. an analytical system which senses belowsaid nozzle.
 145. A flow cytometer system as described in claim 144wherein said first axial motion surface comprises a first axialacceleration surface and wherein said second axial motion surfacecomprises a second axial acceleration surface.
 146. A flow cytometersystem as described in claim 145 wherein said nozzle has accelerationvalues caused by its internal surface and wherein said accelerationvalues are selected from a group comprising: not more than about 0.16m/sec per micron, not more than about 0.05 m/sec per micron away fromthe vicinity of the exit orifice, not more than about 0.10 m/sec permicron away from the vicinity of the exit orifice, not more than about0.13 m/sec per micron away from the vicinity of the exit orifice, notmore than about 0.16 m/sec per micron in the vicinity of the exitorifice, not more than about 0.20 m/sec per micron in the vicinity ofthe exit orifice, not more than about 0.23 m/sec per micron in thevicinity of the exit orifice, not more than about 100×10⁻³ m/sec permicron at a distance of more than 300 um away from the exit orifice, notmore than about 50×10⁻³ m/sec per micron at a distance of more than 300um away from the exit orifice, not more than about 25×10⁻³ m/sec permicron at a distance of more than 300 um away from the exit orifice,such acceleration values with respect to axial location as do notdiscontinuously change along a central axis, not more than about100,000×10⁻⁶ m/sec per micron², not more than about 10,000×10⁻⁶ micron²,not more than about 2,000×10⁻⁶ m/sec per micron², not more than about1,100×10⁻⁶ m/sec per micron², not more than about 100,000×10⁻⁶ m/sec permicron² away from the vicinity of the exit orifice, not more than about50,000×10⁻⁶ m/sec per micron² away from the vicinity of the exitorifice, not more than about 10,000×10⁻⁶ m/sec per micron² away from thevicinity of the exit orifice, not more than about 5,000×10⁻⁶ m/sec permicron² away from the vicinity of the exit orifice, not more than about1,000×10⁻⁶ m/sec per micron² away from the vicinity of the exit orifice,not more than about 300×10⁻⁶ m/sec per micron² away from the vicinity ofthe exit orifice, not more than about 200×10 ⁻⁶ m/sec per micron² at adistance of more than 300 um away from the exit orifice, not more thanabout 100×10⁻⁶ m/sec per micron² at a distance of more than 300 um awayfrom the exit orifice, such rate of change of acceleration values withrespect to axial location as do not discontinuously change along acentral axis, and such rate of change of acceleration values withrespect to axial location as do not change sign along a central axisaway from the vicinity of the exit orifice.
 147. A flow cytometer systemas described in claim 144 wherein said limited maximal accelerationdifferentiation transition area comprises a unitary surface.
 148. A flowcytometer system as described in claim 144 wherein said limited maximalacceleration differentiation transition area comprises a unitary exitorifice.
 149. A flow cytometer system as described in claim 144 whereinsaid analytical system which senses below said nozzle operates at a rateselected from a group comprising at least 500 sorts per second, at least1000 sorts per second, and at least 1500 sorts per second.
 150. A flowcytometer system as described in claim 144 and further comprising apressurization system which operates at least about 50 psi.
 151. A flowcytometer system as described in claim 149 and further comprising asperm collection system.
 152. A flow cytometer system as described inclaim 150 and further comprising a sperm collection system.
 153. A flowcytometer system as described in claims 144, 147, 148, 149, or 150wherein said sample comprises sperm cells selected from a groupcomprising bovine sperm cells and equine sperm cells.
 154. A sexed spermspecimen produced with a flow cytometer system as described in any ofclaims 144, 147, 148, 148, 150, 151, or
 152. 155. A mammal producedthrough use of a sexed sperm specimen produced with a flow cytometersystem as described in any of claims 144, 147, 148, 148, 150, 151, or152.
 156. A flow cytometer system as described in claims 144, 148, 149,150, or 151 and further comprising a single torsional orientation nozzlelocated at least in part below said injection point.
 157. A flowcytometer system as described in claim 156 and further comprising asperm collection system.
 158. A sexed sperm specimen produced with aflow cytometer system as described in claim
 157. 159. A flow cytometersystem as described in claim 158 wherein said sample comprises spermcells selected from a group comprising bovine sperm cells and equinesperm cells.
 160. A mammal produced through use of a sexed spermspecimen produced with a flow cytometer system as described in claim157.
 161. A flow cytometer system as described in claim 160 wherein saidsample comprises sperm cells selected from a group comprising bovinesperm cells and equine sperm cells.
 162. A method of flow cytometrysample processing, comprising the steps of: a. establishing a sheathfluid; b. injecting a sample into said sheath fluid at an injectionpoint; c. subjecting said sample to a first axial motion surface in anozzle; d. transitioning to a second axial motion surface in saidnozzle; e. subjecting said sample to said second axial motion surface insaid nozzle wherein said first and said second axial motion surfacestransition with a maximal acceleration differentiation; f. coordinatingsaid maximal acceleration differentiation so as to not exceed thepractical capabilities of said sample over its length; g. affirmativelylimiting said maximal acceleration differentiation so as to not exceedthe practical capabilities of said sample over its length; h. exitingsaid sample from said nozzle; i. analyzing said sample.
 163. A method offlow cytometry sample processing as described in claim 162 wherein saidstep of subjecting said sample to a first axial motion surface in anozzle comprises the step of subjecting said sample to a first axialacceleration surface and wherein said step of subjecting said sample tosaid second axial motion surface in said nozzle comprises the step ofsubjecting said sample to a second axial acceleration surface whereinsaid first and said second axial motion surfaces transition with amaximal acceleration differentiation.
 164. A method of flow cytometrysample processing as described in claim 162 wherein said nozzle createsacceleration values though its internal surface and wherein saidacceleration values are selected from a group comprising: not more thanabout 0.16 m/sec per micron, not more than about 0.05 m/sec per micronaway from the vicinity of the exit orifice, not more than about 0.10m/sec per micron away from the vicinity of the exit orifice, not morethan about 0.13 m/sec per micron away from the vicinity of the exitorifice, not more than about 0.16 m/sec per micron in the vicinity ofthe exit orifice, not more than about 0.20 m/sec per micron in thevicinity of the exit orifice, not more than about 0.23 m/sec per micronin the vicinity of the exit orifice, not more than about 100×10⁻³ m/secper micron at a distance of more than 300 um away from the exit orifice,not more than about 50×10⁻³ m/sec per micron at a distance of more than300 um away from the exit orifice, not more than about 25×10⁻³ m/sec permicron at a distance of more than 300 um away from the exit orifice,such acceleration values with respect to axial location as do notdiscontinuously change along a central axis, not more than about100,000×10⁻⁶ m/sec per micron², not more than about 10,000×10⁻⁶ m/secper micron², not more than about 2,000×10⁻⁶ m/sec per micron², not morethan about 1,100×10⁻⁶ m/sec per micron², not more than about100,000×10⁻⁶ m/sec per micron² away from the vicinity of the exitorifice, not more than about 50,000×10⁻⁶ m/sec per micron² away from thevicinity of the exit orifice, not more than about 10,000×10⁻⁶ m/sec permicron² away from the vicinity of the exit orifice, not more than about5,000×10⁻⁶ m/sec per micron² away from the vicinity of the exit orifice,not more than about 1,000×10⁻⁶ m/sec per micron² away from the vicinityof the exit orifice, not more than about 300×10⁻⁶ m/sec per micron² awayfrom the vicinity of the exit orifice, not more than about 200×10⁻⁶m/sec per micron² at a distance of more than 300 um away from the exitorifice, not more than about 100×10⁻⁶ m/sec per micron² at a distance ofmore than 300 um away from the exit orifice, such rate of change ofacceleration values with respect to axial location as do notdiscontinuously change along a central axis, and such rate of change ofacceleration values with respect to axial location as do not change signalong a central axis away from the vicinity of the exit orifice.
 165. Amethod of flow cytometry sample processing as described in claim 162wherein said step of transitioning to a second axial motion surface insaid nozzle comprises the step of subjecting said sample to a unitarysurface.
 166. A method of flow cytometry sample processing as describedin claim 162 wherein said step of transitioning to a second axial motionsurface in said nozzle comprises the step of subjecting said sample to aunitary exit orifice.
 167. A method of flow cytometry sample processingas described in claim 162 and further comprising the steps of: a.forming drops around said sample after it has exited said nozzle; and b.sorting said drops at a rate selected from the group comprising at least500 sorts per second, at least 1000 sorts per second, and at least 1500sorts per second.
 168. A method of flow cytometry sample processing asdescribed in claim 162 and further comprising the step of pressurizingsaid nozzle at a pressure of at least 50 psi.
 169. A method of flowcytometry sample processing as described in claim 167 wherein said stepof injecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells into said sheath fluid. 170.A method of flow cytometry sample processing as described in claim 168wherein said step of injecting a sample into said sheath fluid at aninjection point comprises the step of injecting sperm cells into saidsheath fluid.
 171. A method of flow cytometry sample processing asdescribed in claims 162, 165, 166, 167, or 168 wherein said step ofinjecting a sample into said sheath fluid at an injection pointcomprises the step of injecting sperm cells selected from the groupcomprising bovine sperm cells and equine sperm cells into said sheathfluid.
 172. A method of creating a sexed sperm specimen comprising thestep of producing a sexed sperm specimen as described in any of claims162, 165, 166, 167, or
 168. 173. A method of creating a mammalcomprising the step of producing a sexed sperm specimen as described inany of claims 162, 165, 166, 167, 168, 169, or
 170. 174. A method offlow cytometry sample processing as described in claims 162, 166, 167,168, or 169 and further comprising the steps of: a. establishing asingle torsional surface in said nozzle; b. generating single torsionalhydrodynamic forces from said single torsional surface; and c. orientingsaid sample with said single torsional hydrodynamic forces.
 175. Amethod of flow cytometry sample processing as described in claim 174wherein said single torsional hydrodynamic forces and said maximalacceleration differentiation combine and are affirmatively chosen so asto not exceed the practical capabilities of said sample over its length.176. A method of flow cytometry sample processing as described in claim175 wherein said step of injecting a sample into said sheath fluid at aninjection point comprises the step of injecting sperm cells into saidsheath fluid.
 177. A method of creating a sexed sperm specimencomprising the step of producing a sexed sperm specimen as described inclaim 176 and wherein said step of injecting a sample into said sheathfluid at an injection point comprises the step of injecting sperm cellsinto said sheath fluid.
 178. A method of creating a sexed sperm specimencomprising the step of producing a sexed sperm specimen as described inclaim 177 wherein said step of injecting sperm cells into said sheathfluid comprises the step of injecting sperm cells selected from thegroup comprising bovine sperm cells and equine sperm cells into saidsheath fluid.
 179. A method of creating a mammal comprising the step ofproducing a sexed sperm specimen as described in claim 176 and whereinsaid step of injecting a sample into said sheath fluid at an injectionpoint comprises the step of injecting sperm cells into said sheathfluid.
 180. A method of creating a mammal comprising the step ofproducing a sexed sperm specimen as described in claim 179 wherein saidstep of injecting sperm cells into said sheath fluid comprises the stepof injecting sperm cells selected from the group comprising bovine spermcells and equine sperm cells into said sheath fluid.